Flow Cytometry

Flow cytometry is now a widely used method for analyzing expression of cell surface and intracellular molecules, characterizing and defining different cell types in heterogeneous cell populations, assessing the purity of isolated sub populations, and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells. It is predominantly used to measure fluorescence intensity produced by fluorescent-labelled antibodies detecting proteins or ligands that bind to specific cell-associated molecules, such as DNA binding by propidium iodide.

One of the fundamentals of flow cytometry is the ability to measure the properties of individual particles. When a sample in solution is injected into a flow cytometer, the particles are randomly distributed in three-dimensional space. The sample must therefore be ordered into a stream of single particles that can be interrogated by the machine’s detection system. This process is managed by the fluidics system. Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5 μm to 40 μm diameter.

The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. It is widely used in:

Immunophenotyping. In clinical labs, immunophenotyping is useful in diagnosing hematological malignancies such as lymphomas and leukemia.

Cell sorting. The cell sorter is a specialized flow cytometer with the ability to physically isolate cells of interest into separate collection tubes and characterizes each cell.

Cell cycle analysis. can analyze replication states using fluorescent dyes to measure the four distinct phases of the cell cycle and can measure cell aneuploidy associated with chromosomal abnormalities.

Apoptosis. Two distinct types of cell death, apoptosis and necrosis, can be distinguished by flow cytometry on the basis of differences in morphological, biochemical and molecular changes occurring in the dying cells.

Cell proliferation assays. The flow cytometer can measure proliferation by labeling resting cells with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester.

Intracellular calcium flux. The cytometer can monitor the flux of calcium into the cell and measure the extent to which cells respond to the stimuli.